A novel sulfatase, specific for the 3-0 sulfate ester of alpha methyl sulfamino glucopyranoside, has been partially purified from human urine. Using the enzyme as a structural probe, unique 3-0 sulfated glucosamine residues have been located within an octasaccharide fragment of heparin having high affinity for antithrombin. All three isomeric sulfate esters of alpha methyl glucosaminide have now been prepared to provide standards for the identification of sulfated residues in heparin and to define the specificity of the enzyme. In the simple sulfated glucosaminide derivatives, the enzyme is not significantly inhibited by the presence of an additional sulfate group on the 6 position.